Mycoplasma pneumoniae, a member of the class of cell wall-less prokaryotes, Mollicutes, is among the smallest free-living prokaryotes yet described. Furthermore, this species is an important pathogen of the human respiratory tract and the single leading cause of pneumonia in the age group that includes older children and young adults. Despite the unique biological characteristics and taxonomic status of the mycoplasmas and their widespread impact upon public health and agriculture, progress has been slow in applying the tools of modern molecular biology towards a better understanding of the pathogenesis of M,. pneumoniae infections. With recent advances involving transformation and homologous recombination in other mycoplasmas, the genetic analysis of mycoplasma virulence factors is fast becoming directly addressable. Adherence by M. pneumoniae to the respiratory epithelium (cytadherence) is pivotal to successful colonization and disease. The focus of the studies described here is a group of five proteins, designated HMW1-5, the coordinate expression of which correlates with cytadherence phase variation. The long-range objectives of this study are to elucidate how expression of the genes for HMW1-5 is coordinately regulated, to define through genetic means whether each is required for cytadherence, and to clarify the function of each in this process. This project is shifting from the developmental stage involving gene cloning and mapping, to the point where most of the necessary tools are in place to address these objectives directly. The genes for HMW3 and HMW1 have been cloned and mapped and found to be very closely linked. The gene for HMW3 has been sequenced, revealing several unusual features that may reflect protein function. Furthermore, the transposon Tn4001 has been electroporated into M. pneumoniae, conferring resistance to gentamicin and demonstrating the feasibility of transformation studies with M. pneumoniae. In the next project period, specific segments of hmw DNA will be combined with the GmR gene from Tn4001 and electroporated into M. pneumoniae for insertional inactivation of specific hmw genes by homologous recombination. Insertional recombinants will be evaluated for their ability to cluster the adhesin P1 at the attachment organelle, their capacity to cytadhere, and for changes in their protein profile. These studies will be combined with DNA sequencing and mRNA characterization to define hmw transcriptional units and identify at what level hmw gene expression is regulated. Finally, a reporter system will be developed for more detailed evaluation of hmw gene expression.